The present invention relates generally to the fields of Liquid Chromatography (LC), High Performance Liquid Chromatography (HPLC), and preparative chromatography, and comprises a method and apparatus for the accurate and precise measurement of all parameters which affect the quality of the delivery of pressure-driven liquids in an LC or HPLC system. In particular, the invention relates to a method and apparatus for precisely measuring fluid flow rate in such a way as to provide visibility of both short and long term deviations of the flow rate of liquids in LC or HPLC systems.
Liquid chromatography is a widely used technique for separating complex mixtures of chemicals into their constituent parts. This is accomplished by pumping the mixture (mobile phase) through a separation device referred to as a “column.” More specifically, an LC (Liquid Chromatography) column is a cylindrical tube containing a separation media (stationary phase). The separation media is porous so that the mixture will pass through it. As the mixture passes through, the separation media selectively impedes or slows the flow of the various components that make up the mixture. The desired outcome is that those constituents that are less restricted by the separation media exit the column (elute) sooner than more restricted components, which travel at a lower speed through the column.
At its simplest level, an LC system consists of only two components: a pump and an LC column. In practice, however, commercial LC systems often comprise multiple pumps, columns, specialized detectors which are sensitive to the eluents, various fluid handling valves, and sophisticated software for controlling the process and collecting data. It is the mission of many manufacturers to provide sophisticated and consistent LC columns, chemicals, methods, and other necessary equipment in order to achieve consistent separation of the mobile phase into very distinct eluents.
In any LC system, the most fundamental parameter that must be controlled is the flow rate of fluids through the system. The flow rate results from pressure which is applied by the LC pump. It follows in practice that most flow consistency and accuracy issues originate because of the fundamental character of the pump. The majority of LC pumps are cam driven piston pumps, typically having one or two pistons, a fluid inlet, and a fluid outlet. The fluid inlet is typically at or near atmospheric pressure, and the fluid outlet is typically at a high pressure. Outlet pressures can be as high as 5000 PSI, and recently introduced pumps have pushed this limit to approximately 15,000 PSI.
To achieve these pressures, a rotating cam applies force to a high strength piston. Each piston operates in a reciprocating fashion. As the piston moves forward, fluid is pressurized and exits the pump, to be driven through the rest of the LC system. When the piston reaches the end of its stroke, it is retracted as it follows the profile of the rotating cam, and the cylinder is refilled with fluid to be delivered on the next forward stroke. A check (one way) valve is used to keep fluid which has been pushed out of the pump from reentering the pump on the refill stroke. A second check valve keeps fluid which is pressurized by the piston from flowing back out through the pump inlet.
In practice, all piston driven pumps have two intrinsic characteristics that can cause them to provide anomalous deviations in flow rate. It is the common goal of LC system manufacturers to minimize the deleterious effect caused by these characteristics. The first characteristic is visualized by considering a single piston pump. Flow deviation occurs as the piston is being retracted and the cylinder is refilling. During this time, no fluid is delivered from the pump. The lack of additional fluid to compensate for the normal loss of fluid through the LC column causes a momentary pressure drop. This causes the separation and elution processes to slow down until the pump is again delivering fluid at the correct pressure.
Manufacturers offer dual piston pumps as a solution to the above-mentioned anomaly. Ideally, when one piston is refilling, the second piston is delivering fluid, thus eliminating the momentary pressure drop. In practice, a second less apparent anomaly occurs during the changeover from one piston to the other, and is typically masked by the refill pulse in the case of the single piston pump.
The second anomaly is the result of the relationship between the exact moment the outlet check valve(s) open, the desired working system pressure, and a physical characteristic of all fluids, namely, compressibility. Since a check valve opens when the pressure on the inlet side is greater than the pressure on the outlet side, it is necessary to expend some of the piston's stroke to achieve an internal pump pressure equal to the system pressure. This fraction of a stroke is referred to as “pre-compression.” The amount of stroke lost to pre-compression increases as the overall desired system pressure is increased. If the pump or system does not specifically and correctly compensate for the required pre-compression, the flow rate will be incorrect. In other words, a dip or a spike in pressure (and thus flow rate) can result, depending on whether pre-compression compensation is set above or below the required operating pressure to achieve the required flow rate.
With regard to this second anomaly, a very practical consideration is: as an LC column ages, the required pressure to achieve a given flow rate typically increases. Therefore, an LC system that is properly pre-compensated for a new column will suffer degradation in elution consistency if the pre-compensation is not adjusted with regard to the required flow vs. pressure characteristic of the aging column.
A solution to both of these anomalies is available in the form of a pressure reservoir, which is inserted in the hydraulic system at some point between the pump and the LC column. This reservoir stores fluid that is compressed to the operating pressure of the LC column. When a deviation of flow results from the pump, the reservoir absorbs or releases fluid as determined by the size of the reservoir, the actual pressure, and the rate of fluid loss through the column and other hydraulic components. The effect is to lessen the severity of the flow deviation by providing a longer decay time. The pressure decay is exponential, and is according to the formula:P(t)=Ps*e−(t/RC) 
Where:                P(t)=the resulting pressure as a function of time        Ps=the system pressure when the piston refill begins        e=2.71828        t=time        R=resistance to flow or backpressure generated by column to flow        C=capacity in PSI/mL of the pressure reservoir        
In practice, a reservoir is sized to allow an acceptable deviation of pressure at a given operating condition (flow rate and pressure). The trend towards conducting HPLC analysis at lower flow rates is significant with respect to the technique of using a pressure reservoir. The problem lies in attempting to decrease flow rate by simply slowing the rotation of the pump's cam. This approach causes the refill time to be extended inversely with the reduction of flow rate. During refill, system pressure decays exponentially with time. A typical reduction in flow rate will result in a longer period of time in which pressure decay will occur. This allows a system to fall below the flow deviation requirements of the original system design. By way of example, if a pump that is optimized to provide 1% deviation in flow at 100 μL/min is operated at 10 μL/min, a 37% deviation will result. This is true of pumps using mechanically connected cams (driven by a single motor) to drive the pistons, because the refill stroke cannot be altered without affecting the opposing piston's fill stroke. In the case of single piston pumps, there is some ability to increase the speed of the refill stroke and the precision of refill stroke timing by using the inverse of the desired reduction of flow rate.
Other means of driving pistons such as motorized linear screw drives are also available, which can vary the pre-compression appropriately for the fluid and operating pressure. In such case, however, it is still advisable to monitor pulsation performance to verify that the pump has been calibrated and compensated correctly, and is not malfunctioning.
The net effect of the foregoing problem is that eluents do not arrive at the output of the LC column at consistent times, and there is a possibility of the eluents themselves being misclassified. Additional analysis, typically through mass spectroscopy, is often necessary to resolve doubts that occur due to inconsistencies in elution resulting from flow rate fluctuation.
A common method of attempting to correct the aforementioned problem; that is, the inability to simply slow cam-driven pumps to achieve lower flow rates, is called “flow splitting,” or simply “splitting.” Referring to FIG. 3, the topology of a flow splitting system is apparent. In this topology, the pump is operated at a flow rate that is within its proven performance range. The output of the pump drives a three-way “T” type junction. Of the two remaining connections of the T-junction, one is connected to the column; the remaining connection drives a backpressure control device. By adjusting the amount of backpressure on the non-column output of the T, the flow through the column can be controlled.
A serious fundamental problem with this approach, however, is that the backpressure of the column itself varies over time, typically increasing as the column ages. As the column backpressure changes, it is necessary that a corresponding change occur in the regulating output of the T, otherwise column flow will be adversely affected. Even in the presence of either automatic or manual adjustment of the backpressure regulation means, the overall backpressure encountered by the pump changes, potentially shifting its operating point into a pressure range that is undesirable.
As of yet, there is no economical means to characterize column backpressure with regard to flow rate; or to measure the column flow rate on the high pressure side of the column when using a conventional cam-driven pump; or to quantify flow deviation as a function of time and pump operating pressure; or to alert a user as to the presence of adverse operating conditions; or to collect pressure and flow data from an independent device which can be used to validate system performance in the absence of any anomaly.
What is required is an instrument that can be inserted in the fluid path that will report pump pressure, column pressure, fluid temperature and flow data at adequate data rates to observe short term deviations in flow rate. Further, such a system should provide data in a readily readable form to a host PC, Personal Digital Assistant (PDA), laptop, or other programming device, which is capable of capturing and recording data relative to an accurate and independent time base.
Outside the field of the invention, there are many well known methods to determine flow rate, where the rate is comparatively large (>10 mL/minute). These include, for example, the use of turbine wheels, ultrasonic measurement of the velocity of bubbles or solids in a fluid stream, and displacement of a sphere or other indicator suspended within a vertical cone shaped fluid path. None of these methods are viable below sub-mL/minute (e.g. μL/m and nL/m) ranges of flows. Furthermore, none of the methods are suitable to advance the state-of the-art in LC or HPLC because they do not have adequately fast sampling rates to measure short term flow fluctuations.
Historically, liquid flow rates have been measured using a volumetric measuring device (graduated cylinder, pipette, syringe, etc.) and a means of measuring elapsed time. Using the measuring device, an operator typically observes the change in a fluid level or the movement of a trapped bubble to determine the fluid volume that flowed at a given time. The primary disadvantage of this method is that it does not allow adequate time resolution to detect or quantify sufficiently short term deviations in flow. Such deviations typically result from piston pumps and incorrect compensation for compressibility of the liquid being pumped, as has been described hereinabove.
A recently developed method uses two nano-fabricated thermistors placed along the fluid path, and a small heating element positioned near the upstream thermistor. As flow rate increases, there is a proportional increase in the amount of heat which must be applied to attain a constant temperature differential. This “thermal gradient” approach does allow the measurement of short term deviation of flow, but disadvantageously does not allow either a wide dynamic range or optimum insertion of the flowmeter in the flow path due to inability of the thermal gradient type flowmeter to withstand high operating pressures greater than a few hundred PSI.
An additional modern approach uses a pressure drop as a factor in determining flow. A shortcoming of this approach, however, is that the pressure drop is measured across a porous bed (see, e.g., U.S. Pat. No. 6,532,802). Thus the effect of variation of viscosity with temperature is not taken into account. Moreover, the possibility of change in the backpressure characteristic of the flow sensing element is more likely with a porous bed.
The prior art literature and patents describe a variety of methods for remediating flow deviation and pre-compression. However, the drawbacks to the traditional thermal sensing flowmeters illustrate the need for alternatives or improvements in controlling flow deviation and pre-compression in a hydraulics system. These are the primary needs addressed by the present invention.